Functionalization and purification of molecules by reversible group exchange

ABSTRACT

Embodiments of the present disclosure include methods and compositions for functionalizing molecules, such as oligonucleotides, with functional groups, including polyhistidine tags useful in affinity methods. Some embodiments include methods for modifying and purifying complex mixtures of molecules by exchange of functional tags.

The present application is a Continuation of U.S. patent applicationSer. No. 14/717,312 filed May 20, 2015, which is a Continuation of U.S.patent application Ser. No. 13/565,400 filed Aug. 2, 2012, issued asU.S. Pat. No. 9,040,678 on May 26, 2015, which claims priority to U.S.Provisional Patent Application Ser. No. 61/515,663 filed Aug. 5, 2011,which are incorporated herein by reference in their entireties.

BACKGROUND

Immobilized metal ion affinity chromatography (IMAC) is widely used, forexample for protein purification, exploiting the ability of certainamino acid sequences to form a claw-like configuration around theexposed electrons of a metal ion. When polypeptides contain a suitableamino acid motif, such as a series of histidines (polyhistidine), theamino acids of the motif can form coordinate bonds around metal ions,such as Ni²⁺ or Co²⁺, that are present on an IMAC chromatographysurface.

This binding property is particularly useful when a polypeptide ofinterest contains (or is recombinantly engineered to contain) the motif.Even when the polypeptide is in a complex mixture like an expressioncell lysate, the motif can specifically grip the metal ions of the IMACsurface, allowing other components of the mixture to be removed. Becausethe grip of the coordinate bonding can be reversed, the recombinantpolypeptide can then be selectively released from the surface andcollected in a purified state. The motif thus serves as a “purificationtag” for controlled binding, washing and release of the polypeptideusing the IMAC surface. Unlike other chromatography methods that arelimited by the number and expense of specialized affinity groups thatcan be affixed to the matrix, IMAC surfaces can offer bulk densities ofmetal ions, making such methods efficient and scalable.

While generating polypeptides of interest with an attached motif isstraightforward with the tools of recombinant DNA technology, there arepractical barriers to attaching them to other classes of molecules ofinterest, especially when attachment is desired for specific locationson a molecule, or when several molecules are synthesized as a pool andare to be purified in parallel. Thus, IMAC chromatography methods havebeen unavailable for purification and handling of other molecules, forexample oligonucleotides (usually shortened to “oligos”), which areessential for most biotechnological applications. Moreover, there is aneed for methods for obtaining the molecules for use in the purificationmethods, for improved methods for purifying the products, and for theability to manipulate molecules in general by attaching purificationtags and other convenient functional groups by a broadly applicablechemistry.

SUMMARY

Embodiments as disclosed herein relate to methods for modifyingmolecules of interest by exchange of one group of the molecule with adifferent, functionally useful group, using one or more reversiblechemical steps. The molecules of interest can be, for example,biological molecules (biomolecules) such as polypeptides orpolynucleotides, where a group on the molecule is replaced with apurification tag suitable for IMAC purification methods. Functionalizedoligonucleotides are thereby provided with tags such as polyhistidine(His-oligos) and biotin (biotin-oligos) in the form of single oligos orin complex pools.

Some embodiments include methods for generating and enrichingoligonucleotides useful for the purification methods of the disclosure.The products of some of the methods described herein can be used in avariety of applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a method for sequence-specific ligation of twoaldehyde-oligos by using a bridging oligo and a linking reagent.

FIG. 2A depicts a useful exchange moiety, 6-hydrazinonicotinate acetonehydrazone (“Hynic”) and FIG. 2B depicts a bifunctional exchange reagent,His-HyNic-biotin.

FIG. 3 illustrates an embodiment of a method for functionalization offull-length oligos with biotin, while avoiding functionalization ofoligos that are not full-length.

FIG. 4 illustrates particular steps for obtaining a purified pool ofbiotin-oligos.

FIG. 5 depicts the removal of the His tag from a His-oligo using2-pyridine hydrazide aniline.

FIG. 6 depicts an alternate method for producing biotin-oligos.

FIG. 7 depicts a method for using biotin-oligos for targeted enrichment

FIG. 8 depicts overlaid FPLC traces of oligo pool 2 (42 k complexity) atdifferent stages of functionalization. Dotted trace A: starting pool of5′-aldehyde-oligos. Thin trace: pool after exchanging 5′-aldehyde withHis. Thick trace: pool after exchanging 5′-His with biotin.

FIG. 9 depicts a gel electrophoretic analysis of an oligo pool (42 kcomplexity) at different stages of purification using a nickel IMACresin. Lane 1: pool of His-oligos prior to purification on a Ni-column.Lane 2: wash fraction from Ni-column, showing unbound oligos lacking aHis tag. Lane 3: eluate collected from Ni-column, showing purifiedHis-oligos.

FIG. 10 shows a liquid chromatography (LC) elution profile of areference 5′-aldehyde-oligo (95 mer).

FIG. 11 shows the LC elution profile of the oligo after exchange of the5′-aldehyde for His, prior to purification on a Ni-column.

FIG. 12 shows the LC elution profile of the oligos after purification ona Ni-column and exchange of the 5′-His by biotin. The major peak of theLC elution (biotin-oligo, retention time of 9.81 min) was furtheranalyzed by mass spectroscopy (MS), as shown in FIG. 13.

FIG. 13 depicts the MS ion fragmentation pattern and relative ionabundance of the major peak of FIG. 12, demonstrating the relativepurity of the biotin-oligo product.

FIGS. 14A-D show a series of graphs, depicting results discussed inExample 5. FIG. 14A is a bar graph showing the amount of excessbiotinylated oligo remaining in supernatant after streptavidinpull-down. FIG. 14B and FIG. 14C are bar graphs showing that a largespike of biotinylated oligos did not adversely affect the enrichment orcoverage when Exol was used in the assay. FIG. 14D is a bar graphshowing enrichment using a biotinylated oligo pool of 55 k complexity,with and without Exol in the assay.

DETAILED DESCRIPTION

Reversible Chemistry for Exchanging Groups

Embodiments of the present disclosure provide broadly applicable methodsfor modifying molecules that include exchanging one group on a moleculefor a desired functional group, such as a tag for facilitatingpurification of the molecule. After use, the tag can be removed, oroptionally the tag can be further exchanged for a second functionalgroup, such as a useful affinity tag or a label tag, for example. Theconvenient exchange of groups is effected by reversible reactions whereequilibrium conditions are controlled at each stage to direct thereaction forward or reverse as desired. After the functionalization, theaddition of the functional group can be reversed by the same exchangereaction, but under different equilibrium conditions. Moreover, afunctional group added to the molecule can be itself exchanged bysubsequent exchange reactions.

Molecules of Interest

A molecule of interest can be any molecule with an exchangeable groupfor use in embodiments disclosed herein, including inorganic moleculesor molecules of nonbiological origin. Also useful are “biomolecules,”meaning naturally occurring or artificially produced biochemicalmolecules. Molecules include polypeptides, such as proteins or peptides,amino acids, and derivatives thereof; lipids, fatty acids and the like,and derivatives thereof; carbohydrates, complex saccharides (e.g.oligosaccharides, polysaccharides, glycoconjugates, etc.),monosaccharrides and the like, and derivatives thereof; nucleic acids(polynucleotides of any length, including oligonucleotides),nucleotides, nucleosides, purines, pyrimidines and the like, andderivatives thereof; and any other molecules that may be a constituentof a biological sample. Molecules of interest may be a mixed polymer oftwo or more of the various molecules listed above. Other biopolymersinclude known intracellular mediators, co-factors and the like,macromolecular structures and/or assemblies (e.g. cytoskeletal elements,centrioles, chromatin lipid rafts, signal transduction completes), andcytosol.

In some embodiments, a molecule of interest is a different class ofmolecule than many or all of the other molecules in a mixture. Forexample, if the molecule of interest is a nucleic acid, molecules ofdifferent classes would include molecules comprising amino acids,molecules comprising carbohydrates and/or molecules comprising lipids.In other embodiments, a molecule of interest is a different type ofmolecule than many or all of the other molecules in a mixture. Forexample, if the molecule of interest is a nucleic acid having a certainsequence, molecules of different types would include nucleic acidshaving significantly different sequences. An example of significantlydifferent sequences is sequences that have one or more nucleotidesubstitutions at the same or similar nucleotide positions.

Polynucleotides

As used herein, the terms “polynucleotide”, “oligo”, “nucleic acid”, and“nucleic acid sequence” are generally used interchangeably and includesingle-stranded and double-stranded polymers of nucleotide monomers,including 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linkedby internucleotide phosphodiester bond linkages, or internucleotideanalogs, and associated counterions, e.g. H⁺, NH₄ ⁺, trialkylammonium,tetraalkylammonium, Mg²⁺, Na⁺ and the like. The nucleotide monomer unitsmay comprise any of the nucleotides described herein, includingnaturally occurring nucleotides and nucleotide analogs.

Nucleic acids include genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, cRNA,alternatively spliced mRNA, small nucleolar RNA (snoRNA), microRNAs(miRNA), small interfering RNAs (siRNA), piwi RNAs (piRNA), any form ofsynthetic or modified RNA, fragmented nucleic acid, nucleic acidobtained from subcellular organelles such as mitochondria orchloroplasts, and nucleic acid obtained from microorganisms or DNA orRNA viruses that may be present on or in a biological sample.

In nucleic acid sequences described herein, “A” denotes deoxyadenosineor adenosine, “C” denotes deoxycytidine or cytidine, “G” denotesdeoxyguanosine or guanosine, “T” denotes deoxythymidine or thymidine,and “U” denotes deoxyuridine or uridine. For RNA, the deoxyribose isreplaced with ribose in the nucleotide monomers. Nucleic acids may becomposed of a single type of sugar moiety, as in the case of RNA andDNA, or mixtures of different sugar moieties, as in the case of RNA/DNAchimeras.

Nucleic acid sequences herein are generally shown in the 5′-to-3′orientation from left to right, unless otherwise apparent from thecontext or expressly indicated differently. A functional group describedas being in the 5′ direction of a nucleic acid (such as a5′-biotin-oligo) indicates that the group is attached at or near the 5′terminus of a nucleotide or nucleic acid (e.g. directly or indirectlyvia the 5′—O or 5′—OH), rather than at or near the 3′-terminus. Likewisea 3′-functional group is attached at or near the 3′ terminus of thenucleotide or polynucleotide.

Nucleic acids may vary in length and may be intact or full-lengthmolecules or fragments or portions of larger nucleic acid molecules.Nucleic acids can also be partial or full-length copies of nucleic acidsderived from genomic nucleic acids and/or other sources. In particularembodiments, a nucleic acid can comprise at least about 2 nucleotides,at least about 3 nucleotides, at least about 4 nucleotides, at leastabout 5 nucleotides or more than 5 nucleotides. In particularembodiments, a nucleic acid can comprise at least about 5 nucleotides,at least about 10 nucleotides, at least about 20 nucleotides, at leastabout 30 nucleotides, at least about 40 nucleotides, at least about 50nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 150 nucleotides, at least about 200nucleotides, at least about 250 nucleotides, at least about 500nucleotides, or at least about 1,000 nucleotides. In more embodiments, anucleic acid can comprise from about 150 to about 4000 nucleotides, fromabout 500 to about 3,000 nucleotides, or from about 1000 nucleotides toabout 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, 5000nucleotides, 6000 nucleotides, 7000 nucleotides, 8000 nucleotides, 9000nucleotides, or about 10,000 nucleotides in length. Alternatively oradditionally, a nucleic acid can comprise no more than about 100nucleotides, no more than about 250 nucleotides, no more than about 500nucleotides, no more than about 1000 nucleotides, no more than about5000 nucleotides, no more than about 10,000 nucleotides, or no more thanabout 100,000 nucleotides.

As used herein, the term “nucleotide analogs” can refer to syntheticanalogs having modified nucleotide base portions, modified pentoseportions, and/or modified phosphate portions, and, in the case ofpolynucleotides, modified internucleotide linkages, as generallydescribed elsewhere (e.g. Scheit, Nucleotide Analogs (John Wiley 1980);Englisch, Angew. Chem. Int. Ed. Engl. 30:613-29 (1991); Agarwal,Protocols for Polynucleotides and Analogs (Humana Press, 1994); andVerma and Eckstein, Ann. Rev. Biochem. 67:99-134 (1998), all of whichare incorporated herein by reference in their entireties.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

As used herein the term “at least a portion”, “a portion thereof” and/orgrammatical equivalents thereof can refer to any fraction of a wholeamount. For example, “at least a portion” can refer to at least about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9% or100% of a whole amount.

Generally, modified phosphate portions comprise analogs of phosphatewhere the phosphorus atom is in the +5 oxidation state and one or moreof the oxygen atoms is replaced with a non-oxygen moiety such as sulfur.Exemplary phosphate analogs include phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate, andboronophosphates, with any associated counterions, e.g. H⁺, NH₄ ⁺, andNa⁺. Examples of modified nucleotide base portions include5-methylcytosine (5mC); C-5-propynyl analogs including C-5 propynyl-Cand C-5 propynyl-U; 2,6-diaminopurine (also known as 2-amino adenine or2-amino-dA); hypoxanthine, pseudouridine, 2-thiopyrimidine, isocytosine(isoC), 5-methyl isoC, and isoguanine (isoG; see, e.g. U.S. Pat. No.5,432,272, incorporated by reference). Exemplary modified pentoseportions include locked nucleic acid (LNA) analogs including Bz-A-LNA,S-Me-Bz-C-LNA, dmf-G-LNA, and T-LNA (see, e.g. The Glen Report, 16(2):5(2003); Koshkin et al., Tetrahedron Letters 54:3607-30 (1998),incorporated herein by reference in its entirety), and 2′- or3′-modifications where the 2′- or 3′-position is hydrogen, hydroxyl,alkoxy (e.g. methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy,and phenoxy), azido, amino, alkylamino, fluoro, chloro, or bromo.Modified internucleotide linkages include phosphate analogs, analogshaving achiral and uncharged intersubunit linkages (e.g. Sterchak etal., Organic Chem., 52:4202 (1987), incorporated herein by reference inits entirety), and uncharged morpholino-based polymers having achiralintersubunit linkages (see, e.g. U.S. Pat. No. 5,034,506, incorporatedherein by reference in its entirety). Some internucleotide linkageanalogs include morpholidate, acetal, and polyamide-linked heterocycles.In one class of nucleotide analogs known as peptide nucleic acids(including pseudocomplementary peptide nucleic acids (“PNA”)), aconventional sugar-and-internucleotide linkage is replaced with a2-aminoethylglycine amide backbone polymer. See, e.g. Nielsen et al.,Science, 254:1497-1500 (1991); Egholm et al., J. Am. Chem. Soc.,114:1895-1897 (1992); Demidov et al., Proc. Natl. Acad. Sci. USA99:5953-58 (2002); Nielsen (ed.), Peptide Nucleic Acids: Protocols andApplications (Horizon Bioscience 2004), all of which are incorporatedherein by reference in their entireties.

Molecules from Biological Samples

Molecules useful in disclosed embodiments can be provided by a varietyof means, such as by purification from biological samples. As usedherein, a “biological sample” can refer to a plurality of molecules thatinclude a molecule of interest. In some embodiments, a biological samplecan comprise a library or mixture of compounds. Biological samples canbe derived from biological and non-biological origins. For example,biological samples can include a blood sample, biopsy specimen, tissueexplant, organ culture, biological fluid or any other tissue or cellpreparation, or fraction, derivative, or isolate therefrom, from asubject or a biological source. The subject or biological source may bea human or non-human animal, including mammals and non-mammals,vertebrates and invertebrates, and may also be any other multicellularorganism or single-celled organism such as a eukaryotic (includingplants and algae) or prokaryotic organism, archaeon, microorganism (e.g.bacteria, archaea, fungi, protists, viruses), aquatic plankton, aprimary cell culture or culture adapted cell line including geneticallyengineered cell lines that may contain chromosomally integrated orepisomal recombinant nucleic acid sequences, immortalized orimmortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell lines,stem cells, germ cells (e.g. sperm, oöcytes) and the like. For example,nucleic acids may be obtained from primary cells, cell lines, freshlyisolated cells or tissues, frozen cells or tissues, paraffin embeddedcells or tissues, fixed cells or tissues, and/or laser-dissected cellsor tissues. In certain embodiments, the nucleic acids may be derived,purified, or isolated from any known prokaryotic or eukaryotic organismor virus.

Synthetic Polynucleotides of Interest

Methods for providing synthetic nucleic acids are well known in the art.For example, in some methods, nucleoside phosphoramidites can beutilized. Such nucleoside phosphoramidites are examples of monomerreagents that may be utilized with the methods, from biological samples.In some embodiments, nucleoside phosphoramidates include derivatives ofnatural or synthetic nucleosides in which protection groups (sometimesreferred to a blocking groups) are added to reactive exocyclic amine andhydroxyl groups, and in which an N,N-diisopropyl phosphoramidite groupis attached to the 3′-hydroxyl group of each nucleoside. Examples ofprotecting groups include acid-labile dimethoxytrityl (DMT) groups.

Nucleic acids can also be provided as products of chemical or enzymaticamplification reactions, such as a polymerase chain reaction (PCR). Ifdesired, amplification products can be produced having an exchangeablegroup (such as an aldehyde) by using forward or reverse primersincorporating the exchangeable group so that amplicons are producedhaving the exchangeable group.

In some synthesis methods, full-length products can be modified forexample by an additional final group (such as by a formylindolephosphoramidite modifier during synthesis) to serve as a terminatinggroup, as well as a useful means for distinguishing full-length fromincomplete synthesis products.

Functional Tags

The present disclosure provides molecules of interest that have groupsthat can serve as functional tags. In some embodiments, a functional tagcan include a chemical and/or biological moiety that provides one ormore desired functional characteristics.

The functional tags described herein can be present at any location of amolecule of interest. In some embodiments, a molecule of interestcomprising a polypeptide or protein can include a functional tag at theC-terminal end, at an intermediate amino acid, or at the N-terminal end.In other embodiments, a molecule of interest comprising a nucleic acidmay include a functional tag at the 5′ end, at an intermediatenucleotide, or at the 3′ end of the nucleic acid.

For example, a functional tag can be an aldehyde group present on anoligo of interest, such as on the 5′—OH or the 3′—OH, or as part of alarger moiety, such as a formylindole. Other examples of functional tagsinclude label tags and affinity tags, which can serve as purificationtags.

Label Tags

As used herein, the term “label tag” refers to any identifiable tag,label, or group. Many different species of label tags can be used in theembodiments herein, individually or in combination with one or moreother label tag species. Examples of label tags include fluorophores,radioisotopes, chromogens, enzymes, antigens including epitope tags,semiconductor nanocrystals such as quantum dots, heavy metals, dyes,phosphorescence groups, chemiluminescent groups, electrochemicaldetection moieties, binding proteins, phosphors, rare earth chelates,transition metal chelates, near-infrared dyes, electrochemiluminescencelabels, and mass-spectrometer-compatible reporter moieties, such as masstags, charge tags, and isotopes.

In certain embodiments, a label tag can emit a signal, which can befluorescent, a chemiluminescent, a bioluminescent, a phosphorescent, aradioactive, a calorimetric, or an electrochemiluminescent signal, forexample. Other label tags include spectral labels such as fluorescentdyes (e.g. fluorescein isothiocyanate, Texas red, rhodamine),radiolabels (e.g. ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P), enzymes (e.g.horse-radish peroxidase, and alkaline phosphatase) spectral calorimetriclabels such as colloidal gold or colored glass or plastic (e.g.polystyrene, polypropylene, latex) beads; magnetic, electrical, thermallabels; and mass tags. Label tags can also include magnetic particles.More label tags include 1- and 2-aminonaphthalene,p,p′-diaminostilbenes, pyrenes, quaternary phenanthridine salts,9-aminoacridines, p,p′-diaminobenzophenone imines, anthracenes,oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin, porphyrins,triarylmethanes and flavin. Individual fluorescent compounds that havefunctionalities for linking a label to a molecule of interest includedansyl chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthydrol;rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene;N-phenyl 2-amino-6-sulfonatonaphthalene;4-acetamido-4-isothiocyanato-stilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate;N-phenyl-N-methyl-2-aminoaphthalene-6-sulfonate; ethidium bromide;stebrine; auromine-0,2-(9′-anthroyl)palmitate; dansylphosphatidylethanolamine; N,N′-dioctadecyloxacarbocyanine:N,N′-dihexyloxacarbocyanine; merocyanine, 4-(3′-pyrenyl)stearate;d-3-aminodesoxy-equilenin; 12-(9′-anthroyl)stearate; 2-methylanthracene;9-vinylanthracene; 2,2′-(vinylene-p-phenylene)bisbenzoxazole;p-bis(4-methyl-5-phenyl-2oxazolyl))benzene;6-dimethylamino-1,2-benzophenazin; retinol; bis(3′-aminopyridinium)1,10-decandiyldiiodide; sulfonaphthylhydrazone of hellibrienin;chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-(p-(2benzimidazolyl)-phenyl)maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin; 4-chloro7-nitro-2,1,3-enzooxadiazole;merocyanine 540; resorufin; rose bengal; 2,4-diphenyl-3(2H)-furanone,fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, rhodamine, tetramethylrhodamine, eosin,erythrosin, coumarin, methyl-coumarins, quantum dots (also referred toas “nanocrystals”: see U.S. Pat. No. 6,544,732, hereby incorporated byreference in its entirety), pyrene, Malachite green, stilbene, LuciferYellow, Cascade Blue™, Texas Red, Cyanine dyes (such as Cy3 and Cy5),Alexa dyes, phycoerythin, bodipy, and others described in Haugland etal. (eds.), Handbook of Fluorescent Probes and Research Chemicals (6thed. 1996), expressly incorporated by reference herein. More examples oflabel tags include, but are not limited to, affinity labels such asbiotin, avidin, streptavidin, digoxigenin, antibody Fc domain, proteinA, protein G, antibody antigen-binding domain, and lectins.

Label tags can be selected for inclusion in the presently disclosedcompositions and methods depending on the particular parameter orparameters that may be of interest for particular molecules inbiological samples in particular applications. Examples of parametersthat may be detected by some label tags include detection of thepresence of one or more of an amine, an alcohol, an aldehyde, water, athiol, a sulfide, a nitrite, avidin, biotin, an immunoglobulin, anoligosaccharide, a nucleic acid, a polypeptide, an enzyme, acytoskeletal protein, a reactive oxygen species, a metal ion, pH, Na⁺,K⁺, Cl⁻, a cyanide, a phosphate, selenium, a protease, a nuclease, akinase, a phosphatase, a glycosidase, and a microbial contaminant.

Affinity Tags

Some embodiments disclosed herein include methods for isolating amolecule of interest. In some such methods, a molecule of interestcomprises a purification tag that contacts a binding partner. Theassociation of the purification tag and binding partner may be used toseparate the molecule of interest from a mixture of molecules.

A purification tag can comprise moieties with the same or similarstructures. In certain embodiments, the tagging moiety of an affinitytag can be associated with a functional tag directly by a single bond orvia a linkage of stable chemical bonds, in linear, branched or cyclicarrangements, optionally including single, double, triple bond, aromaticcarbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogenbonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygenbonds, phosphorus-nitrogen bonds, and any combination thereof. Incertain embodiments, the association between the tagging moiety andfunctional tag comprises ether, thioether, carboxamide, sulfonamide,urea or urethane moieties. In preferred embodiments, the linkagecomprises a polyalkylene chain, i.e., a linear or branched arrangementof carbon-carbon bonds. In other embodiments, the linkage comprises apolyalkylene oxide chain, including a polyethylene glycol moiety.

Examples of affinity tags include, but are not limited to, biotin,digoxigenin (Dig), dinitrophenol (DNP), zinc fingers, fluorinatedpolymers, and polypeptide sequences such as polyhistidine motifs.

Affinity Substrates

Some embodiments disclosed herein can be used with solid substrateshaving an affinity for a functional group. In some embodiments, thebinding partner may be immobilized on an affinity substrate. As usedherein the term “affinity substrate” can refer to an immobile matrix orsupport bound to a binding partner that is capable of forming a strongand preferably reversible interaction with the purification tag of amolecule. An affinity substrate can include a resin, a bead, a particle,a membrane, a gel. The binding partner recognizes or binds to thepurification tag specifically. Specific binding partners will depend onthe affinity tag, but include charged moieties and one member of abinding pair such as receptor-ligand, antibody-antigen,carbohydrate-lectin, and biotin-streptavidin.

Preferably, the purification tag binds to the affinity substrate with anaffinity constant (K_(a)) of at least about 10³ M⁻¹, 10⁴ M⁻¹, preferablyat least about 10⁵ M⁻¹, more preferably at least about 10⁶ M⁻¹, stillmore preferably at least about 10⁷ M⁻¹ and even more preferably at leastabout 10⁸ M⁻¹ or stronger affinity. Affinity constants may be determinedusing well known techniques including any number of standard bindingassays or techniques, for example, those described by Scatchard et al.,Ann. N.Y. Acad. Sci. 51:660 (1949); and R. K. Scopes, ProteinPurification: Principles and Practice (Springer-Verlag 1987,incorporated by reference herein in their entireties).

IMAC Resins

In certain embodiments, the binding partner comprises a metal ion boundto an immobile matrix via coordination bonds which can be useful in ionmetal affinity chromatography (IMAC). Due to the availability of metalions to the solution phase, the binding capacities of IMAC resins can beunusually high. The amount of IMAC substrate can be controlled to scaleup a method disclosed herein, or to serve as a control on the amount ofproduct yield, if desired. IMAC surfaces are available in variousformats (bulk slurries, pre-loaded columns, spin columns, coated 96-wellplates, magnetic beads for example) and the term “resin” is used hereinto mean any of these formats. The convenient format of IMAC resins isamenable to highly parallel formats and automation for processing manyindividual molecules or pools of molecules.

Examples of metal ions that have specific recognition capability forpurification tags, such as polyhistidines, include Ni²⁺ and Co²⁺.Suitable IMAC resins include commercially available kits such as NiSepharose® (Amersham Bioscience), Ni-NTA-agarose (Qiagen), His60 NiSuperflow™ (Clontech Laboratories, Inc.), HisPur™ cobalt or nickelresins (Pierce Chemical Co.), and TALON® His-Tag purification resin(Clontech) to name a few.

A molecule of interest can have one or more, for example, two or moredifferent affinity tags (such as a His tag and a biotin) and theaffinity substrate having one or more corresponding binding agents (suchas a bifunctional IMAC resin having both metal ions and streptavidin forthe exemplary His and biotin tag, respectively). Such an exemplarybifunctional combination of binding affinities allows much more specificbinding in the methods of the disclosure. As discussed above IMAC resinshave found use in protein purification methods, but the presentdisclosure provides methods for using IMAC resins for purification ofany molecule of interest having an exchangeable group. Moreover, in someembodiments a molecule of interest may have two or more, three or more,four or more, etc. affinity tags which are the same, for example are thesame functionally, such as two or more biotins, two or more His tags,and the like. Further, if there are a plurality of affinity tags (e.g.,two or more, etc.), there could be a disproportionate amount of thedifferent tags, for example more biotin tags than His tags thandigoxigenin tags, for example.

As used herein, the terms “purifying,” “isolating,” “separating,” (andtheir grammatical forms) are used interchangeably and refer tosubstantially or essentially enriching a mixture for the molecule ofinterest. In some embodiments, enriching a mixture for a molecule ofinterest includes removing components (collectively “contaminants”) of amixture other than a molecule of interest. In certain embodiments,“isolating a nucleic acid” may refer to separating an oligo (e.g. afull-length oligo) from other oligos (e.g. truncated oligos, fragmentedoligos, etc.) in a reaction mixture following a chemical or enzymaticoligo synthesis. In other embodiments, “isolating a nucleic acid” may bepurifying a nucleic acid from the nucleotide sequences that flank it ina naturally occurring state, such as a DNA fragment that has beenseparated or removed from the sequences that are normally adjacent tothe fragment. In yet other embodiments, “isolating a molecule” may referto extracting a molecule from a cell, tissue, or organism such that itis no longer present in the cell, tissue or organism in its naturalstate. It will be appreciated that purifying does not require that themolecule of interest be completely separated from all contaminants, butcan refer to increasing the proportion of one or more particularmolecules in a mixture of molecules relative to contaminants, includingpurification so the molecule is substantially free of contaminants.

As used herein, “substantially free of contaminants” refers tocompositions having less than about 75% contaminating molecules, lessthan about 50% contaminating molecules, less than about 25%contaminating molecules, less than about 20% contaminating molecules,less than about 15% contaminating molecules, less than about 10%contaminating molecules, less than about 5% contaminating molecules,less than about 4% contaminating molecules, less than about 3%contaminating molecules, less than about 2% contaminating molecules,less than about 1% contaminating molecules.

Non-full-length Oligonucleotides

Some methods provided herein are advantageous in purifying full-lengthsynthesized oligos, especially those synthesized by stepwise addition ofmonomeric units. For example, in nature (and in many older chemicalmethods), polynucleotides are typically synthesized in the 5′-to-3′direction. However, a particularly useful method for obtaining syntheticoligonucleotides is stepwise synthesis in the 3′-to-5′ direction. Forsome synthetic methods, stepwise addition can result in a mixture offull-length nucleic acids (e.g. n-mer) and nucleic acids shorter thanfull-length, due to inefficiencies during individual synthetic steps.These can include side products resulting from incomplete deprotectionor modification, or protecting groups cleaved from the nucleotide basesafter synthesis. Other undesirable side products result from incompletesynthesis that are less then full-length (e.g. (n−1)-mer, (n−2)-mer,(n−3)-mer, etc).

Oligos can be synthesized by methods such as phophoramidite synthesissuch that only full-length oligos incorporate a terminal nucleotidecomprising an aldehyde group. However, the final incorporation is notnecessarily 100% efficient, and the number of oligos having the aldehydegroup can be, for example 60%, 70%, 80%, or 90%.

When the functional tag is added to a polymer as the first or last stepof full-length synthesis, the absence of the tag can serve as anindicator that the synthesis was not complete for that polymer (e.g.FIG. 3, 210 a, 210 b, 210 c). The presence of the tag indicates that thepolymer is full-length, as exemplified by the aldehyde-oligo (200) inFIG. 3. The tag can further function as a means of attachment for easiermanipulation.

Solid Phase Attachment

In some embodiments, oligos can be attached to a solid phase surface forease of manipulation, for example in performing chemical reactions onthe oligos, washing, and collecting the oligos by detachment from thesurface. Oligos can be attached to surfaces, such as beads, by variousmethods known in the art. Alternatively, aldehyde-oligos used indisclosed embodiments can be attached to a solid phase through thehydrazide reactions discussed in more detail below. In methods disclosedherein, solid phase attachment can be either reversible or irreversible.For example, molecules of interest (e.g., oligonucleotides) can beattached to a solid phase and can be released through reversiblechemistries such as released by cleaving a cleavable linker incorporatedinto a molecule of interest. Further, a molecule of interest could beitself cleaved. Cleavable linkers that could be incorporated intomolecules of interest such as oligonucleotides include, but are notlimited to, azide groups, alkoxy groups, disulphide groups, diol groups,diazo groups, ester groups, sulfone azide, alyl or silyl ether groups,acid labile groups, Sieber groups, indole groups, t-butyl Sieber groups,electrophilically cleavable groups, nucleophilically cleavable groups,photocleavable groups, cleavage groups that cleave under reductiveconditions or oxidative conditions, cleavage via use of safety-catchgroups, cleavage by elimination mechanism and metal assisted cleavagegroups. Conversely, a molecule of interest could be irreversibly affixedto solid phase supports, such as through covalent binding of themolecule to a substrate either directly or indirectly (e.g., via alinker, etc.). Irreversible affixation of a molecule of interest to asubstrate is particularly advantageous for high-capture efficiencies.

In other embodiments, an affinity tag can be irreversibly coupled to asubstrate but still comprise a reversible moiety and/or be complexedwith a linker that comprises a reversible aspect and/or a molecule ofinterest that comprises a reversible aspect, or a combination thereof.For example, a molecule of interest could be irreversibly affixed to asolid substrate via, for example, covalent attachment. For example, insome embodiments, an affinity tag comprises irreversible couplingchemistry that irreversibly binds to a solid support, but additionallythe affinity tag comprises a reversible sequence (e.g., a cleavablesequence) that allows for release of the molecule of interest from thecoupling chemistry. In other embodiments, a linker that links theaffinity tag to the molecule of interest comprises a reversiblesequence, for example a cleavage group that can be cleaved to releasethe molecule of interest from the permanently affixed couplingchemistry. In other embodiments, the molecule of interest itselfcomprises releasable sequences, such as cleavage groups, that can becleaved to release the molecule of interest from the irreversiblyaffixed coupling chemistry on the solid support. In some embodiments, anaffinity tag/linker/molecule of interest complex can comprise aplurality of irreversible and reversible sequences which can be used,for example, for purification purposes of the molecule of interest froma sample.

A skilled artisan will understand the myriad options available for bothreversibly and irreversibly binding a molecule of interest to asubstrate.

Reversible Attachment of 5′-aldehyde Oligos to a Solid Phase

An alternate method for attaching aldehyde-oligos is provided in thisdisclosure, which enables aldehyde-oligos to be attached or detachedfrom a solid surface by forming a reversible imine (generallyR¹R²—C═N—R³) linkage between an aldehyde-oligo and anamine-functionalized surface. Any combination of functionalities thatcan form an imine bond can be used. For example, an amine can be on theoligo and the aldehyde can be at the surface. The aldehyde can be also aketone. The amine functionality can be a simple aliphatic amine or ahydrazine. A useful amine functionality is an alkoxyamine, sincealkoxyamine-based imines can form a stronger bond than aliphatic-basedimines.

In one embodiment a full-length oligo is provided having a 5′-aldehydemoiety. Where the oligo is provided in the form of a 3′-to-5′ synthesisreaction product, the final addition can be an aldehyde-containingmoiety, so that incomplete synthesis products (non-full-length) lack thealdehyde. The amino-surface can be provided by reaction of3-aminopropyltrimethoxy silane with a glass surface, e.g. glass beads, aflat glass microscope slide, controlled pore glass (CPG), or aminefunctionalized polystyrene beads. The aldehyde-oligo is allowed to reactwith the solid-phase amine, for example in a citrate buffer (pH 6.5), toform a stable imine bond that anchors the oligo to the solid phase. Thesolid support can be washed to remove any unbound oligo, such asnon-full-length synthesis products that lack an aldehyde.

Optionally, the bound oligo can be cleaved from the solid support usingacid-catalyzed hydrolysis conditions to regenerate the full-lengthaldehyde-oligo and the free amine-surface. The oligo can also be cleavedby transamination with an aqueous solution of para-toluenesulfonic acid(p-TsOH or tosylic acid), which can be catalyzed by aniline or scandium(III) trifluoromethanesulfonate (scandium triflate). Yet anothercleavage method involves washing the solid support with anamino-functionalized molecule so that the aldehyde transaminates withthe non-bound amine, forming an imine on the free aldehyde. It should benoted, however, that this last embodiment does not restore the aldehydefunctionality.

The reversible attachment/detachment of oligos to solid surfaces isparticularly useful for flow-through synthesis methods. The oligo issynthesized on the amidite-functionalized CPG, cleaved from the CPG withammonium hydroxide, then immediately neutralized to pH 6, so theoligo-synthesis product is allowed to flow onto the amine-functionalizedCPG. Non-full-length oligos do not attach to the amine-CPG and arewashed away. If desired, the full-length oligos can be detached andcollected for further use.

Bridge Ligation of Aldehyde-oligonucleotides

Another use for aldehyde-oligos is the sequence-specific ligation of twofunctionalized oligos to obtain a longer oligo containing a definedcombination of sequences. The functionalization can be an aldehyde,although the two oligos can be biotin-oligos, as provided byembodiments, for example those found below.

A specific embodiment is exemplified in FIG. 1, where the first oligo isprovided with an aldehyde at the 3′ terminus. The second oligo isprovided with an aldehyde at the 5′-terminus. As disclosed herein, oneor both oligos are optionally immobilized to a solid substrate (such asa bead) to facilitate handling, washing, and collection. Either one orboth oligos can also contain additional sequences for use in variousapplications (such as genotyping, identification, and sequencingreactions) as long as the additional sequences do not interfere with thelinking reaction.

By way of example, a bridging oligo is also provided that contains (atits 3′ terminus) a first sequence that is complementary to the 3′portion of the first oligo. The bridging oligo also contains (at its 5′terminus) a second sequence that is complementary to the 5′ portion ofthe second oligo. The bridging oligo preferably contains sequence thatis sufficient in length to allow sequence-specific hybridization tofirst and second oligos under similar conditions. The first and secondsequences of the linker are allowed to hybridize to the first and secondoligos, forming a sequence-specific hybridization complex where the twoaldehydes are brought into proximity.

A bifunctional linking reagent is provided that comprises at least twomoieties that could react with aldehyde groups, such as a hydrazine oramine moiety. A hydrazine-aldehyde reaction can be catalyzed with acatalyst of aniline and its derivatives. Other useful moieties include,but are not limited to, maleimide-thiol, maleimide-amine,streptavidin-NHS (N-hydroxysuccinimide), isothiocyanate-amine,amine-carboxyl catalyzed by EDC, thiol-thiol, and dialkene (e.g.obtained by a water-soluble methathesis catalyst). A particular reactivealdehyde linker (Aldrich 639958) is

The aldehyde-reactive moieties of the linking reagent are allowed toreact with the adjacent aldehydes of the two oligos of the hybridizationcomplex, forming a covalent linkage between the two oligos. Since thehybridization complex can form when the bridging oligo specificallycoordinates the combination of the first and second oligos, the ligationcan occur only for the two designated oligos of interest.

This method is particularly useful for combinatorial applications usinga pool of various first oligos (e.g., with different sequences, such asa set of DNA bar codes) in combination with a pool of various secondoligos (e.g., with different sequences, such as an orthogonal set of DNAbar codes, a recognition or detection sequence, or a biologically activesequence). The method allows generation of oligos having a desiredcombination of first and second oligo sequences by introducingrelatively short bridging oligos to coordinate the chemical ligation ofdifferent combinations of first and second oligos. For instance, poolsof 100-mer first oligos and 400-mer second oligos can be joined togenerate specific 500-mers by means of a bridging oligo having two20-base complementary sequences for specific hybridization. If desired,the ligated oligos can be used while attached to a solid phase, ordetached for further use. The skilled artisan will understand that thefirst and second sequences of the resulting oligos are separated by thecovalent linker, so not all biochemical processes (such as polymeraseamplification) are feasible across the linker. However, the linker doesnot affect applications where the first and second sequences need onlybe present on the same molecule, such as when oligos (and attachedbeads) are identified by independent binding events. Thus, methoddisclosed herein provides a rapid and convenient method for generating acombinatorial library of beads having a predefined selection ofsequences.

Exchange Reagents

A general method for exchanging groups uses “exchange reagents” thatcomprise a functional group (as discussed herein) attached to the restof the molecule by a bond, allowing the functional group exchange withanother group on the molecule of interest. If desired, the amount ofexchange reagent used can be limited to control the total yield of thefinal product. According to the application and stage of synthesis, thebond can be irreversible or reversible as desired. An example of anirreversible exchange reagent is a biotin oxo-amine, which appears inthe middle portion of FIG. 4.

Examples of reversible exchange reagent are nucleophiles, for example areagent containing an imine (═C═N—), where the imine serves as a stable,but reversible bond. These reagents can be formed by reaction withhydrazides (generally (R¹(C═O)R²—N—N—R³R⁴), hydroxylamine (NH₂OH) andthe like. Useful examples include hydrazones (R¹R²C═N—NH₂) and theirderivatives, such as bisarylhydrazones, which are formed between abenzaldehyde and a hydrazinopyridine group. Schiff bases (e.g.R¹R²C═N—R³ or HR²C═N—R³) are also useful imine reagents. Yet anotherreversible reagent is hydrazinic acid (hydrazine with an acyl group). Inother embodiments, the exchange reagent contains an electrophile, aslong as the reagent can form a reversible bond.

The exchange reagent can contain an exchange moiety to facilitate theexchange. A particularly useful exchange moiety is 6-hydrazinonicotinateacetone hydrazone (“HyNic”) (Solulink, Inc., San Diego), shown in FIG.2A, where the arrow indicates the imine bond. A modified version ofHyNic can also be used where the dimethyl hydrazone (—NH—N═C(CH₃)₂) isreplaced by a hydrazine (—NH—NH₂), thus 6-hydrazinonicotinamide (alsoreferred to herein as HyNic). Other modified versions include replacingthe dimethyl hydrazone with a nucleophile such as methyl hydrazone(—NH—N═CH(CH₃)) or (—NH—N═CH₂). A particular exchange reagent can have aHyNic moiety and a functional group to be exchanged. For example thefunctional group can be any purification tag such as a polyhistidine,such as a His tag coupled to the exchange reagent via a peptide bond atthe N-terminus of the His tag. An exemplary exchange reagent isHis-HyNic, where R₁ in FIG. 2A is 6 histidine residues. Another usefulexchange reagent is biotin-HyNic. Yet another useful affinity tagreagent comprises both a purification tag and a functional group: FIG.2B shows a His-HyNic-biotin reagent that couples with analdehyde-labeled oligo to generate an oligo labeled with both His (H₆)and biotin.

Reversible Chemistries

One advantage of using a hydrazide group is it can reversibly form theimine bond (—C═N—) when coupled to an aldehyde group to form arelatively stable Schiff base. Other reversible chemistries can also beused in the disclosed method. For example, an imine can be reversiblyconverted to a ketone or aldehyde, which can be reversibly converted toan enamine. Another example is 2-cyclohexenone, which can be reversiblyconverted (using CH₃NH₂) to a β-amino ketone product.

Yet another reversible chemistry that can be used in methods disclosedherein are the various “click chemistry” reactions (e.g. U.S. Pat. Nos.6,737,236 and 7,427,678, each incorporated herein by reference in itsentirety). A useful family of reactions is the azide alkyne Huisgencycloaddition, which uses a copper catalyst (e.g. U.S. Pat. Nos.7,375,234 and 7,763,736, each incorporated herein by reference in itsentirety). Other reactions include copper-free Huisgen reactions(“metal-free click”) using strained alkynes.

The reversible bond is used for exchanging a group on a molecule ofinterest for a desired functional group, such as a purification tag. Theconvenient exchange of groups is effected by reversible reactions whereequilibrium conditions are controlled at each stage to direct thereaction forward or reverse as desired. Even if thermodynamicallydisfavored, a reaction can be driven forward by providing excessreactant or by withdrawing product as it is formed (for example bysequestration using phase transition) to allow the reaction to proceedcontinuously. When desired, the same reaction can be driven in reverseby providing excess product or withdrawing reactant. The manipulation ofthe direction of reversible reactions at will is thus exploited toexchange functional groups in a chain of reactions to yield a finalproduct.

Functionalization of Molecules with His Tags by Exchange

In the methods provided herein, the molecule of interest comprises anexchangeable group. An exchange reagent is provided, which comprises afunctional group attached to an exchange moiety. The molecule ofinterest is contacted with the exchange reagent under conditions and fora time sufficient for the imine bond to be broken between the exchangemoiety and the functional group, and for a new imine bond to form,bonding the functional group to the molecule of interest. Thus, theexchangeable group is replaced with the functional group on the moleculeof interest.

Where the molecule of interest in an oligo, the oligo can be single- ordouble-stranded, and its strandedness can be maintained (e.g., using theappropriate hybridization or denaturation conditions) to obtain single-or double-stranded oligo products.

An embodiment of the exchange reaction is exemplified in FIG. 3 and FIG.4, where the molecule of interest is an oligo with an exchangeablealdehyde group (200). The exchange reagent is His-HyNic, which containsan imine, and the exchange moiety is HyNic, and the functional group ispolyhistidine (abbreviated His₆, H₆ or simply His), which can serve as apurification tag in later steps. After the exchange reaction, the Hisbecomes covalently linked to the oligo via a hydrazone (═C═N—NH—) toprovide a His-oligo (220). However, oligos that lack the exchangeablealdehyde, such as incompletely synthesized products (210 a, 210 b, 210c) are not functionalized and lack a His tag. The exchange reaction thuscan result in a mixture of full-length His-oligos and non-taggedincomplete products (e.g., contaminants).

IMAC Chromatography

As discussed herein, a particular affinity substrate is an IMACchromatography surface, such as a chelated nickel resin. The mixturecomprising the full length His oligos as exemplified above is allowed tobind to a nickel resin, and the His-tagged molecules are bound to theNi-column, whereas the untagged incomplete products are washed off.Where the molecule of interest is an oligonucleotide, the wash steps canbe made unexpectedly stringent compared to conventional wash steps whenpurifying polypeptides. For example, a wash of NaOH or urea woulddenature proteins bound to an affinity substrate, but it does notsignificantly affect bound oligonucleotides or the polyhistidine motif,and can serve to remove proteinaceous contaminants such as nucleases orany undesirable Ni-binding proteins that may occur naturally in thesample.

The bound His-tagged molecules can be eluted from the Ni-column, forexample by competitive elution with metal ions in solution.

If desired, methods can include a step that comprises releasing thefunctional tag (e.g., by cleavage, etc.) to yield a purified molecule.In the example of the hydrazide reaction, an affinity tag can bereplaced by reversing the formation of the imine bond via reactionthrough aldehyde as described herein, while introducing a non-functionalmoiety in place of the affinity tag. As shown in FIG. 5, for example,the His-HyNic moiety that is coupled to a molecule can be replaced by anon-functional hydrazide moiety by incubating with 2-pyridine hydrazideaniline. The His tag is thus cleaved to generate a substantially purebiotinylated oligo. A His tag can also be removed by endopeptidases,which do not affect oligos, for example. Alternatively, the additionreaction of the functional group can be such as reversed by the sameexchange reaction, but under different equilibrium conditions.

Further Exchanges

In certain exchange reactions, the product remains in an exchangeablestate that can be exploited for further manipulation, taking advantageof the functionalization.

Accordingly, the products of the exchange reaction or the purificationstep can be used in one or more subsequent exchange reactions. Forexample the functional group can itself be exchanged for a secondfunctional group to yield another product, and so forth. In FIG. 3,after obtaining His-oligos (220), a second exchange reagent ofbiotin-HyNic is used, where HyNic is the exchange moiety and the biotinis the second functional group. The His-tag of the His-oligo is thusreplaced with the biotin group to yield a biotin-oligo. Alternatively,the second exchange reagent can be methoxyamine to yield an unlabeledoligo product.

Irreversible Exchanges for End Product

In other embodiments, it can be advantageous to obtain a final productby using a nonreversible exchange at one or more steps. Thus, a methodcan involve at least one initial exchange (using at least one reversiblereaction), followed by a nonreversible exchange so that the finalproduct does not readily revert to an earlier intermediate product. As aresult, the final product is obtained in higher yields and with greaterstability. A nonreversible exchange step can be facilitated for exampleby a nucleophile, such as an oxo-amine (—O—NH₂), between oligo andfunctional group (see, e.g. West and Otto, Current Drug DiscoveryTechnologies 2(3):1144-1153 (2005), incorporated herein by reference inits entirety).

As exemplified in FIG. 4, the His-oligos can be reacted with biotinoxo-amine to exchange the His tag for a biotin tag, yieldingbiotin-oligos. Any residual His-oligos can be separated frombiotin-oligo products by passing the mixture through a nickel resin asecond time to yield pure biotin-oligos, for example.

Advantages

The simple binding and wash steps of the methods lend themselves toautomation. Where the affinity substrate is a chromatography resin inthe form of beads, the methods disclosed herein can be readily scaledup, taking advantage of the high binding capacity of IMAC surfaces tofunctionalize and purify mmol quantities of oligos. Another benefit ofthis approach is that the relatively low cost of components like HyNicand IMAC resins, which can be readily regenerated after use.

Another significant advantage to methods disclosed herein is that thesteps are relatively unaffected by the overall size of the molecule ofinterest, such as the length of polymers. When the molecules are longsynthetic oligos, gel electrophoresis and HPLC methods are unable tocleanly separate full-length products (e.g., X bases) from incompleteproducts (X-1 bases, X-2 bases, etc.). Thus, where purification ofX-mers is difficult to achieve by conventional methods, the presentdisclosure provides a method for purifying only full-length molecules.Thus, the method is highly tolerant to oligo pools of varying quality.

Surprisingly, the method can be applied to complex pools tofunctionalize, in the same step, complex pools of molecules of interest.Conventional methods (such as gel electrophoresis or HPLC) are notpractical for purifying mixtures en masse.

More Methods for Making Biotin-oligonucleotides

Some embodiments of the methods provided herein include methods formaking biotin-oligos entirely in liquid phase. Oligos are providedhaving a 5′-phosphate and a 3′-terminal block (3′-blocked oligo). Theblock can be a dideoxynucleotide or another phosphate (both of which canbe added via commercially available phosphoramidites). An oligo having afunctional group (5′-functional oligo) at the 5′ terminus is alsoprovided, such as an aldehyde-oligo, as long as the functional group isnot a hydroxyl or phosphate. The 3′-blocked oligo and 5′-functionaloligo are ligated to generate an oligo having 5′-functional group andthe 3′-block. As an example, FIG. 6 depicts a 3′-blocked oligo(5′-phosphate-oligo-dideoxynucleotide-3′) and a 5′-functional oligo(5′-biotin-oligo). The oligos are ligated with T4 ligase to form afull-length 5′-biotin-oligo-dideoxynucleotide-3′ species that can beused in the other methods provided herein. If desired, the 3′-block canbe removed by standard techniques.

As synthesis reactions to provide the 3′-blocked oligo (“major species”)are not necessarily 100% efficient, the starting oligos may containincomplete oligos that lack the 5′-phosphorylation or the3′-dideoxynucleotide as undesired synthesis failures or “minor species”.To remove these minor species from the reaction product, FIG. 6 furtherdemonstrates that oligos with free 5′-hydroxyls can be phosphorylatedwith a kinase such as T4 polynucleotide kinase (PNK). The5′-phosphorylated minor species can then be degraded by including anexonuclease in the reaction, such as lambda exonuclease, whichpreferentially digests 5′-phosphorylated oligos. To remove any residual,unreacted 5′-functional oligos, an exonuclease, such as E. coliexonuclease I can be further included in the reaction. Any remainingfree nucleotides can be removed by simple ethanol precipitation,dialysis or size-exclusion chromatography. Thus, full-lengthbiotin-oligos, unaffected by the exonucleases, can be produced by themethod.

Use of Biotinylated Probes in Targeted Enrichment

When working with samples containing complex mixtures of molecules, suchas genomic DNA, it can be useful to “pull out” target DNA sequences ofparticular interest, thereby selectively enriching the sample for thetargeted sequences. In some methods of targeted enrichment,sequence-specific oligos are provided that are labeled with haptens,such as biotin. The oligos can be anchored (via streptavidin, forexample) to a solid phase surface to serve as capture probes. When thecapture probes anneal to the DNA sequences of interest, they can bepulled out from the complex mixture by means of the solid phase, forexample easily handled paramagnetic beads.

For complete binding of biotinylated capture probes, it is advantageousto provide streptavidin beads in stoichiometric excess. As thecomplexity of the probe pool increases, however, the amount ofstreptavidin beads required for stoichiometric excess becomesprohibitive. This can be especially difficult when there are a largenumber of biotin-labeled probes that have not annealed to a target DNA,yet still compete for binding to streptavidin beads. Removal of excessbiotin probe greatly reduces the amount of streptavidin beads requiredin the assay.

In one embodiment, excess probes can be removed by incubating the duplexprobe-target mixture with exonuclease I, which digests single-strandedDNA from the 3′ terminus (FIG. 7). The desired target:probe complexescan be protected by various methods provided herein. In anotherembodiment, phosphorothioate nucleotides can be added to the 3′ terminusor body of the target library DNA. In yet another embodiment, blockingoligos can be annealed at the 3′ end of the target DNA molecule tocreate 3′ duplexes that are resistant to exonuclease I digestion. Theseblocking oligos are also useful to prevent target-target interactionsleading to reduced enrichment. The method is highly scalable, asdemonstrated in Example 5.

The following Examples provide illustrative embodiments and do not inany way limit the scope of the methods and compositions provided herein.

EXAMPLES Example 1 Synthesis and Purification of His-tagged Oligos

The following example describes the preparation of highly complex poolsof 5′-aldehyde-labeled oligos. The 5′-aldehyde groups of the oligos wereexchanged for His tags using reversible chemistry. The His-tags wereuseful for purifying the oligos on Ni-resins.

Synthesis of 5′-aldehyde oligos

Oligos with a 5′-aldehyde functional group were synthesized on an oligosynthesizer (Illumina, Inc., San Diego Calif.) at an average yield of 10nmol per oligo. Individual oligos were normalized for concentration andcombined in pools of about 42,000 complexity in 0.1×TE buffer. In otherexperiments, pools of 1,000 and 12,000 complexity were used.

Eight separate pools were prepared having varying ranges of % GC: Pool 1(15-37% GC), Pool 2 (37-42%), Pool 3 (42-46%), Pool 4 (46-49%), Pool 5(49-50%), Pool 6 (50-55%), Pool 7 (55-61%), Pool 8 (61-88%). For the 42K pools, 300 ml was precipitated with cold ethanol/sodium acetate. Thepellet was washed once with 25 ml cold 70% ethanol and dried overnightin a vacuum oven at room temperature. The pellet was redissolved in 150ml of 1 M citrate buffer, pH 6.0.

For more detailed mass spectroscopy analysis of the process, a singlereference oligo (about 95 bp) was also obtained, having a 5′-aldehydefunctional group (TriLink Biotechnologies, San Diego Calif.). The 8pools of oligos and the single reference oligo were processed in asimilar manner as described below.

Preparation of 5′ His-labeled Oligos by Exchange

The 5′-aldehyde oligos were quantified by optical absorbancespectroscopy on a NanoDrop UV-Vis instrument (Thermo Fisher Scientific)to be a total of 30 μmol. Next, 10M urea was added, bringing theconcentration of the oligo to 1 mM. A solution of 0.66 gHyNic/PEG2/Hexa-His reagent (Solulink) in 1 ml 1M citrate buffer, pH 6.0was prepared and added to the oligo mixture, resulting in a 20:1 molarexcess of His-reagent:oligo. Next, 2.1 g of neat aniline was added for afinal aniline concentration of 100 mM. The exchange mixture incubated ona rotisserie apparatus for 1 hour at room temperature. The oligos wereprecipitated with cold ethanol/sodium acetate, washed three times withcold 70% ethanol, and then vacuum-dried at room temperature for 4 hours.The pellets were redissolved in pre-warmed HPLC-grade water, yieldingunpurified 5′-His-labeled oligos.

Purification of 5′-His-labeled Oligos

Nickel chromatography resin beads having up to 60 mg/ml protein-bindingcapacity (His60 Ni Superflow Resin, Clontech) was prepared as 200 ml ofpre-homogenized slurry in two separate 225 ml centrifuge tubes, andcentrifuged at 1200 rfu for 5 min (Eppendorf 5810 benchtop centrifuge).The supernatant was removed by aspiration and discarded; the resin waswashed with 100 ml 8M urea in 5×PBS buffer, pH 7.4. The washing wasrepeated three times to equilibrate the resin.

After equilibration of the Ni resin slurry, a 25 ml aliquot of theunpurified 5′-His-labeled oligo product was added to each centrifugetube. An additional 75 ml of 5×PBS buffer containing 8M urea was addedto each tube, and the tube was placed on the rotisserie at roomtemperature for at least 16 hours. After incubation, the tubes werecentrifuged at 1200 rfu for 5 min at room temperature. The supernatantwas removed by aspiration and discarded. The Ni resin was transferred toa plastic Büchner vacuum filter flask and washed with 500 ml WashBuffer: 1×PBS containing 20 mM imidazole. Next, the resin was washedwith 20 ml of 0.01N NaOH and subsequently washed with Wash Buffer. Thewashed Ni resin was divided into two aliquots and transferred to fresh225 ml centrifuge tubes.

To each tube was added 100 ml Elution Buffer: 500 mM imidazole and 1×PBSbuffer (optionally with 10 mM DTT). The tubes were placed on therotisserie for 5 min and centrifuged for 5 min at 1200 rfu. The liquidwas aspirated and collected. The Ni resin was then transferred to aBüchner vacuum filter flask and washed, in parts, with 500 ml of 500 mMimidazole solution and 1×PBS buffer. The collected washings wereconcentrated to about 15 ml using a Centricon 70 (10 kDa MWCO)centrifugal dialysis assembly (Millipore PN UFC701008). The solution waswashed with 4×50 ml HPLC-grade water, concentrating the His-tagged oligoproduct after each wash with the Centricon centrifugal dialysis assemblyand concentrated to about 20 ml. A 10 μl aliquot of the concentratedsolution was quantified using the Nanodrop instrument. Of the solutionobtained from the single reference oligo, a 20 μl aliquot of a 10 μMsolution was analyzed by LC-MS, and another 10 μl aliquot was analyzedby FPLC.

Preparation of 5′ Biotin-labeled Oligos by Exchange

The His-tagged oligo product was transferred to a new 225 ml centrifugetube. Then, 150 ml of 1M citrate buffer at pH 6.0 and 150 ml of 10M ureasolution were added. A molar excess (e.g. 5- to 10-fold) of biotin-oxoamine (biotin-aldoxime, Aldehyde Reactive Probe, Dojindo MolecularTechnologies, PN A305-10) was added relative to the amount of oligo,e.g. for 30 μmol of total oligo, 150 μmol to 300 μmol of the biotinoxo-amine was added. Alternately, biotin-HyNic (Solulink) orbiotin-hydrazide was used (Thermo Scientific, Pierce research products),for example at 5- to 15-fold molar excess. Subsequently, 2.7 g ofaniline was added, and the exchange mixture was incubated on arotisserie at room temperature for 4 hours. The oligos were thenprecipitated using cold ethanol/sodium acetate, the pellet washed with3× with cold 70% ethanol, and the pellet was dried in a vacuum ovenovernight at room temperature. The pellet was redissolved in pre-warmed50 ml of HPLC-grade water.

A 50 ml solution of the biotinylated oligos were contacted with the Niresin as described above, but the washings were collected and retained.The washings were concentrated using a Centricon 70 centrifugal dialysisapparatus as described above, and washed 4×50 ml with HPLC-grade waterdown to a final concentration of about 25 ml in HPLC-grade water. A 10μl aliquot of the final purified solution was quantified with opticalabsorbance spectroscopy on a Nanodrop instrument, and for LC-MS and FPLCanalyses. The final yield was 7.5 μmol, for a total process yield of25%. The His- and biotin-oligos can be stored and were stable for atleast 6 months.

Example 2 Analysis of His-oligo Pools by FPLC

FPLC analysis was performed with an Äkta Explorer FPLC system (GEHealthcare) fitted with a nickel-resin cartridge (HisPur Ni-NTAChromatography Cartridge, Pierce Biotechnology), using a method similarto that described by the column vendor for FPLC analysis of His-labeledmolecules. The signal of the oligos was monitored at 280 nm. The sampleswere prepared as 50 μL volume in water at 20 μM concentration. The flowrate was set at 0.2 ml/min. The binary solvent profile was 100% Buffer Auntil after the non-Histidine-containing oligos were eluted (approx. 25min), then 100% Buffer B to elute off the His-oligos. Buffer A consistedof 50 mM Phosphate, pH 7.4, 20 mM imidazole; 300 mM NaCl. Buffer Bconsisted of 50 mM Phosphate, pH 7.4, 300 mM imidazole, 300 mM NaCl.

The chemical transformation, purity, and yield of the pools wereestablished by capturing the FPLC trace signal (FIG. 8). The reportedyields and purities were validated by using pure His-oligos as inputmaterial. Overall, a purity was obtained of 97-99% (pool 1-6), 95% (pool7), 90% (pool 8).

Analysis by gel electrophoresis demonstrated that after Ni-columnpurification, impurities such as truncated oligos were efficientlyremoved. Moreover, a single band for the His-tagged product was observed(FIG. 9, lane 3). A minor reduction in purity was observed going fromlow to high percent GC pools. For example, pool 1 had a purity of 95%,while pool 8 had a purity of 85% using FPLC analysis.

Example 3 Analysis of Reference Oligo by LC-MS

Mass spectrometry was performed on the single reference oligo by liquidchromatography—mass spectrometry (LC-MS) (Novatia, LLC). The oligo wasfound to incorporate the His tag at the aldehyde site after the firstreaction, and then exchange the biotin functionality for the His tagafter the second reaction. Furthermore, the LC-MS analysis of thepurified oligo found no starting material, no His-tag intermediateproducts, and no synthesis truncation anomalies of the finalbiotinylated product. FIG. 10 shows an LC-MS trace of the 95 bp oligowith a 5′-aldehyde modifier. 9.29 min. peak corresponds to the5′-aldehyde oligonucleotide (Expected MW: 29888, Found: 29888). FIG. 11shows an LC-MS trace of non-purified 95 bp oligo with the 5′-hexa-Hismodifier. 9.67 min peak corresponds to the 5′-hexa-His oligonucleotide(Expected MW: 31006, Found: 31006). FIG. 12 shows an LC-MS trace of theprocessed 95 bp oligo with the 5′-biotin functionality. 9.81 min peakcorresponds to the 5′-Biotin oligonucleotide (Expected MW: 30390, Found:30390). FIG. 13 shows an MS expansion of the 9.81 min retention timepeak, demonstrating the high purity of the biotinylated product.Expected MW: 30390, Found: 30390).

Example 4 Improvement of Binding and Elution with Urea

Several steps were identified to further improve the binding and elutionof the target oligos. Improvements included the addition of urea duringthe Ni-column binding step; addition of a slurry of His-oligonucleotideto Ni-Resin with an increased binding time of up to 4 hours; addition ofurea in the exchange reaction; and the addition of 0.01 N NaOH wash.Each of these steps was found to either improve yield or increasepurity. For example, the addition of urea in the exchange reactionimproved yields by 20%-70%. An additional advantage of urea is theprevention of undesirable cross-hybridization between intermediate oligoproducts, such as two His-oligos.

The reported yields were validated using pure His-oligonucleotides asinput material. The purity and labeling degree was established bycapturing the FPLC trace signal. Additional confirmation that the finalpurified biotin-oligonucleotides were biotin labeled was obtained byperforming a streptavidin-shift assay with the biotin-modifiedoligonucleotides, then analyzing by gel electrophoresis. Measuring thereduction in gel migration with excess streptavidin (versus nostreptavidin) showed that all products shift completely upon addition ofstreptavidin. The results are summarized in Table 1.

TABLE 1 Pool/fraction (%) Aldehyde-labeled oligonucleotides have“non-oligo” with ~50% OD₂₆₀ absorbance: aldehyde-oligos: ~60% yield forHis-to-biotin exchange: >90% streptavidin gel-shift assay confirmsbiotinylated: >95% Purity of pool 1-6: 97-99% Purity of pool 7: 95%Purity of pool 8: 90% Overall yield per pool: ~25%

The above method was performed on a single 95 bp oligonucleotide with analdehyde group at the 5′ end. At particular stages in the method, theoligonucleotide was analyzed using liquid chromatography-massspectrometry (LC-MS; Novatia LLC). The oligos were found to incorporatethe His-tag at the aldehyde site after the first reaction, and thenexchange the biotin functionality for the His-tag after the secondreaction. Furthermore, the LC-MS showed no starting material, no His-tagintermediate products, and no synthesis truncation anomalies. Theresults are depicted in FIG. 10.

The results showed that crude oligonucleotide loading as high as 150μmol per column were readily processed, resulting in product recovery ofgreater than 50% and purity of greater than 95%.

Example 5 Use of Biotin-oligos for Targeted Enrichment of Samples

This example demonstrates a scalable, targeted enrichment method usingbiotin-oligos and exonuclease I. An enrichment pool of about 2500different biotinylated oligos was prepared. A separate pool was preparedwith exogenous biotinylated oligo non-complementary to any of the targetlibrary elements. Excess biotinylated oligos were spiked into a 2.5 k(2500) complexity enrichment oligo pool at varying levels to mimic 10 k,60 k, 100 k, 200 k, and 400 k total oligo complexity.

FIG. 14A is a bar graph showing the amount of excess biotinylated oligoremaining in supernatant after streptavidin pull-down. The excess oligolimited the effective capture of biotinylated probe-target duplexes.Without Exol treatment, a large excess of biotinylated oligo remained.With Exol treatment (Exol+), however, the amount of excess biotinylatedoligo was reduced by up to 100,000-fold (5 logs). FIG. 14B and FIG. 14Cdemonstrate that a large spike of biotinylated oligos did not adverselyaffect the enrichment or coverage when Exol was used in the assay. Thissuggests the enrichment assay can be scaled up to 300,000 loci (humanexome scale). FIG. 14D shows enrichment using a biotinylated oligo poolof 55 k complexity, with and without Exol in the assay. The inclusion ofExol greatly improved the enrichment efficiency.

The headings and subheadings used herein are only for readingconvenience and are not intended to define or limit the scope of thepresent disclosure. The present disclosure describes severalcompositions and methods that are susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theembodiments disclosed herein. Consequently, it is not intended that thisdisclosure be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention.

All references cited herein including, but not limited to, published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

What is claimed is:
 1. A method for modifying a nucleic acid, the methodcomprising: (a) contacting a sample comprising a nucleic acid with abinding partner immobilized on an affinity substrate, wherein thenucleic acid comprises a purification tag, under conditions to form afirst covalent bond coupling the nucleic acid to the binding partner;(b) separating the substrate-coupled nucleic acid from the sample,thereby isolating the nucleic acid from the sample; and (c) performingan exchange reaction comprising contacting the nucleic acid isolated instep (b) with an exchange reagent under conditions sufficient to breakthe first covalent bond between the nucleic acid and the binding partnerand to form a second covalent bond between the exchange reagent and thenucleic acid.
 2. The method of claim 1, wherein the first covalent bondis a —C═N— bond.
 3. The method of claim 1 or claim 2, wherein thepurification tag is an aldehyde and the binding partner comprises anamine moiety.
 4. The method of claim 3, wherein the amine moiety is amoiety comprising a —NHNH₂ group.
 5. The method of claim 3, wherein the5′ terminal nucleotide of the nucleic acid comprises the aldehydepurification tag.
 6. The method of claim 1, wherein the binding partneris irreversibly associated with the substrate.
 7. The method of claim 1,wherein the substrate is a bead, a magnetic bead, a paramagnetic bead, aresin, a particle, a magnetic particle, a membrane, or a gel.
 8. Themethod of claim 1 or claim 2, wherein the second covalent bond is a—C═N— bond.
 9. The method of claim 1, wherein the exchange reagentcomprises an —O—NH₂ group.
 10. The method of claim 1, wherein theexchange reagent is hydroxylamine.
 11. The method of claim 1, whereinthe exchange reagent comprises an —O—NH₂ group and a functional tag. 12.The method of claim 11, wherein the functional tag is selected from thegroup consisting of histidine, biotin, glutathione-S-transferase, afluorophore, a radioisotope, a chromogen, an enzyme, and an epitope. 13.The method of claim 12, wherein the functional tag is biotin.
 14. Themethod of claim 1, wherein the separating does not comprise highperformance liquid chromatography.
 15. The method of claim 1, whereinthe nucleic acid comprises DNA.
 16. The method of claim 1, wherein thesample comprises a plurality of synthesized nucleic acids comprisingfull-length synthesized nucleic acid molecules and partially synthesizednucleic acid molecules.